Irigenin, a novel lead from Iris confusa for management of Helicobacter pylori infection with selective COX-2 and HpIMPDH inhibitory potential

The development of new natural drugs for Helicobacter pylori (H. pylori) management has recently received significant attention. Iris confusa (I. confusa) was long used for the treatment of bacterial infections and gastritis. This study aimed at evaluating its effect on management of H. pylori infection and exploring its bioactive metabolites. The inhibitory potential of the polar (PF), non-polar (NPF) fractions and the isolated compounds against H. pylori using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in addition to their cyclooxygenases (COX-1 and COX-2), and nitric oxide (NO) inhibitory activities were assessed. The most biologically active compound was tested for its selective H. pylori inosine-5′-monophosphate dehydrogenase (HpIMPDH) inhibitory potential. Chromatographic purification of PF and NPF allowed isolation of tectoridin, orientin, irigenin, tectorigenin, isoarborinol and stigmasterol. The PF exhibited significant anti-H. pylori (MIC 62.50 µg/mL), COX-1, COX-2 (IC50 of 112.08 ± 0.60 and 47.90 ± 1.50 µg/mL respectively, selectivity index SI of 2.34), and NO (IC50 47.80 ± 0.89 µg/mL) inhibitory activities, while irigenin was the most potent isolated compound. Irigenin was found to have a promising activity against HpIMPDH enzyme (IC50 of 2.07 ± 1.90 μM) with low activity against human hIMPDH2 (IC50 > 10 μM) than clarithromycin, assuring its selectivity. Overall, I. confusa and its isolated compounds may serve as a potential source of plant-based drugs for H. pylori control. This study scientifically validated the claimed anti-bacterial activity of I. confusa and revealed irigenin potential as a novel lead exhibiting anti H. pylori activity in a first record.

Major constituents isolation. The PF and NPF were screened on pre-coated silica gel 60 F 254 using solvent system S 1 (methylene chloride:methanol:formic acid 85:15:0.2 v/v/v) and S 2 (methylene chloride:methanol 97:3 v/v), respectively. The spots were examined in UV light before and after ammonia vapour exposure and AlCl 3 spraying and after being sprayed with p-anisaldehyde/H 2 SO 4 followed by heating at 110 °C. PF and NPF purification using a vacuum liquid chromatography column (VLC) packed with silica gel H 60, ion exchange resin (diaion HP-20), sephadex LH 20 and silica gel 60 were illustrated in detail in Fig. S1. Similar fractions were pooled together and evaporated to dryness under reduced pressure yielding compounds P 1 (yellow powder, R f = 0.28, S 1 ), P 2 (yellow powder, R f = 0.18, S 1 ), P 3 (yellow crystals, R f = 0.69, S 1 ) and P 4 (yellow crystals, R f = 0.55, S 1 ) from the PF and N 1 (white crystals, R f = 0.62, S 2 ) and N 2 (white crystals, R f = 0.43, S 2 ) from the NPF.
Determination of anti-Helicobacter pylori activity. The anti-Helicobacter pylori activity of the PF and NPF as well as the isolated compounds was evaluated against H. pylori ATCC 700392 (type strain, obtained from the American Type Culture Collection unit (ATCC), using microwell dilution method and clarithromycin as standard drug 37 . The detailed procedures were described in the supplementary file.
Determination of COX-1 and COX-2 inhibitory activity. The tested samples and the standard drugs (ibuprofen and celecoxib) were prepared in dimethyl sulfoxide and subsequent eight twofold dilutions (125-0.98 µg) were carried out in a 96-well plate. The inhibitory COX activity was assayed colourimetrically by examining the inhibition of the ovine COX-1 and human recombinant COX-2 enzymes as described by George et al. 38 , using a COX inhibitor screening assay kit. The detailed procedures were described in the supplementary file.
Determination of nitric oxide (NO) inhibitory activity. The macrophage cell line, RAW 264.7 was obtained from Vaccines, Sera and Drugs Egyptian Company (VACSERA). It was cultured in (RPMI, 1640) medium supplemented with 10% fetal bovine serum and 1% gentamicin 39 . The detailed procedures were described in the supplementary file.
In vitro HpIMPDH inhibition assay. The most potent isolated compound, as revealed from the previous assays, was screened at different concentrations (10-0.078 µM) in triplicates. The assay was carried out according to Galal 40 and was described in detail in the supplementary file.
In vitro hIMPDH2 inhibition assay. Human inosine-5′-monophosphate dehydrogenase (hIMPDH2) was purchased from NovoCIB SAS (Lyon, France). It was used for the in vitro screening of the most potent isolated compound, as revealed from the previous assays, against H. pylori and the standard drug at the concentrations (10-0.078 µM) in triplicates 40 . The detailed procedures were described in the supplementary file.
Quantitative determination of the main phytochemical classes and antioxidant assay. Total phenolic content (TPC). Determination of TPC was carried out according to the European Pharmacopeia procedure 41 , using the Folin-Ciocalteu colourimetric method. It depends on measuring the intensity of the blue colour produced in correlation to the reducing power of existing phenolics. Determinations were carried out in triplicates; results are the mean values ± standard deviations and expressed as μg gallic acid equivalent (GAE) per mg dry fraction (μg GAE/mg).
Total flavonoid content (TFC). TFC was determined based on measuring the intensity of the yellow colour developed upon reaction of flavonoids with aluminium chloride reagent 42 . Quercetin was used to compute the standard calibration curve. Determinations were carried out in triplicates; results were the mean values ± standard deviation and expressed as μg quercetin equivalent (QE) per mg dry fraction (μg QE/mg).
Total triterpene content (TTC). TTC was determined based on measuring the intensity of the red-purple colour developed upon reaction of perchloric acid-oxidized triterpenes in glacial acetic acid with vanillin 43 . Ursolic acid was used to compute the standard calibration curve. Determinations were carried out in triplicates; results were the mean values ± standard deviations and expressed as μg ursolic acid equivalent (UAE) per mg dry fraction (μg UAE/mg).
DPPH assay. The DPPH anti-oxidant assay was carried out as described by Romano et al. 44 with some modifications. The PF and NPF of I. confusa underground parts were separately dissolved in methanol by the aid of sonication to give a set of serial dilutions for each sample. Experiments were performed in triplicates using a 96-wells plate. The reaction mixture in each case consisted of 22 μL of the tested sample and 200 μL of 0.004% DPPH in methanol. The non-quenched DPPH radicals were assessed spectrophotometrically at λmax = 492 nm using a micro-plate reader.
Ethical statement. Collection

Results
Purification of PF and NPF. Chromatographic purification of I. confusa underground parts PF allowed the isolation of three isoflavonoids; tectoridin (P 1 ), irigenin (P 3 ), and tectorigenin (P 4 ), and one flavone, orientin (P 2 ). On the other hand, purification of the NPF led to the isolation of one triterpene, isoarborinol (N 1 ), and one sterol, stigmasterol (N 2 ). The isolated compounds (P 1 -P 4 and N 1 -N 2 ) were identified through their physicochemical characters, spectroscopic analysis, UV spectral data and via comparing their 1 H and 13 C NMR data with the published data (Tables S1-S4 in the supplementary file). Their structures are shown in Fig. 1.
The isolated compounds showed higher inhibitory potential against COX-2 than COX-1 enzyme. Among all tested samples, irigenin showed the highest selectivity for COX-2 (SI = 3.26).

IMPDH inhibition assays.
On the basis of the previous in vitro assays on I. confusa underground parts PF and NPF as well as their isolated compounds (Table 1), the most active compound was irigenin. Thus, irigenin was selected to test its HpIMPDH inhibitory activity by monitoring NADH production at concentration range of 10-0.078 µM compared to clarithromycin as standard drug. Also, to examine irigenin selectivity toward the bacterial IMPDH, human hIMPDH2 inhibition potential was assayed. Results showed that irigenin (P 3 ) exhibited potent inhibitory potential against HpIMPDH enzyme with IC 50 of 2.07 ± 1.90 μM ( Table 2). Whereas the IC 50 value of clarithromycin (standard drug) was 0.51 ± 0.58 μM. In addition, irigenin was less active and safer against hIMPDH2 with IC 50 > 10 μM, compared with clarithromycin (IC 50 3.10 ± 2.10 μM), which make sure that its selectivity is toward HpIMPDH in contrast of clarithromycin. I. confusa underground parts PF exhibited high DPPH scavenging activity with EC50 of 41.68 ± 6.67 μg/mL. The polar fraction TPC amounted 99.05 ± 0.02 μg GAE/mg dried fraction and TFC amounted 98.31 ± 0.04 μg QE/mg dried fraction while, the non-polar fraction TTC amounted 126.30 ± 0.04 μg UAE/mg dried (Table 3).

Discussion
The eradication of H. pylori infection has been proven to prevent gastric or duodenal relapses. Direct correlation was observed between the development of gastric adenocarcinoma and the long-term infection with H. Pylori. Therefore, antibiotics such as clarithromycin have been used for treatment. The declining eradication rates provoked the search for alternative therapeutic options which should be more effective and safer. Exploring medicinal plants along with their phytoconstituents for H. pylori infection control is not just a way to discover safer pharmaceutical alternatives, but also is a trial to discover a natural affordable effective drug especially in developing countries. The selection of Iris in this study was based on our previous experience with this genus diverse metabolites content and antibacterial potential 29,35 . As well as, I. confusa has been used a long time ago in the treatment of bacterial infections and gastritis in traditional Chinese medicine 33 . Hence, the in vitro H. pylori, COX-1, COX-2, and NO inhibition potency of I. confusa underground parts PF, NPF, and their isolated compounds were assessed. Additionally, the selective IMPDH inhibition activity of the most potent isolated compound, irigenin, was examined.
Purification of the PF led to the isolation of four metabolites (P 1 -P 4 ) while two compounds (N 1 -N 2 ) were purified from the NPF. Compounds P 1 -P 4 spectral data were in agreement with the reported data of tectoridin 45 , orientin 46 , irigenin 47 , and tectorigenin 45 . Compounds N 1 -N 2 spectral data were in agreement with the reported    49 . This is the first report for the presence of isoarborinol (N 1 ) in genus Iris. P 1 -P 4 and N 2 are herein isolated from I. confusa for the first time. I. confusa underground parts PF and its isolated compounds showed significant inhibitory effects on H. pylori. Irigenin was superior over tectorigenin as anti-H. pylori. This was attributed to the presence of methoxy group at C-4′ in irigenin, which increases the H. pylori inhibitory effect than the hydroxyl group in C-4′ of tectorigenin as previously reported by Park et al. 50 .
Although tectorigenin (P4) exhibited certain inhibitory activity against H. pylori, its corresponding O-glycoside; tectoridine (P1) exhibited no activity. This was attributed to the effect of glycosylation at 7-OH of tectoridin which caused dramatic decrease in the H. pylori inhibitory activity compared to its aglycone tectorigenin. This dramatic decrease in the activity of the isoflavone glycosides was well documented before 50 . The observed anti-H. pylori activity of orientin matched that previously reported by Król-Kogus et al. 51 .
The inflammation usually associated with H. pylori infection drained the authors interest to explore the COX-1 and COX-2 inhibitory potential. The need to explore drugs with selective COX-2 inhibitory potential that are able to decrease PGs dependent inflammation while maintaining protective gastric mucosal PGs synthesis intact has increased in recent decades. Finding COX-2 inhibitors, but not specific and still having a degree for COX-1 inhibition 18,52 was the main target.
Nitric oxide is free oxygen radical and has cytotoxic effect in pathological processes, particularly in inflammatory disorders. Nitric oxide is a potent proinflammatory molecule secreted during inflammation and causes vasodilation and cellular migration. At higher concentrations, it downregulates adhesion molecules and induces apoptosis of inflammatory cells. Inhibition of NOS (inducible nitric oxide synthase) is beneficial for the treatment of inflammatory disease [53][54][55][56] . Irigenin exhibited significant potential in inhibiting nitric oxide followed by isoarborinol.
Many microbial infections are characterized by rapid proliferation that is supported by guanine nucleotide pool expansion in the rapidly dividing cells. Inosine 5ʹ-monophosphate dehydrogenase (IMPDH), an important enzyme required for the new synthesis of guanine nucleotides, is an interesting target for antimicrobial drug development 57 . This enzyme, IMPDH, catalyzes the oxidation of inosine 5ʹ-monophosphate (IMP) to xanthosine 5ʹ-monophosphate (XMP) leading to reduction of nicotinamide adenine dinucleotide (NAD + ), which is an important step in guanine nucleotides de novo synthesis. IMPDH inhibition surely leads to major fall in guanine nucleotide pool which consequently blocks proliferation 57 .
The most active anti H. pylori and anti-inflammatory tested compound; irigenin (P 3 ) was chosen to test its inhibitory potential against the bacterial HpIMPDH enzyme and to evaluate its selectivity relative to the host enzyme (hIMPDH2). It was found to have promising activity against the HpIMPDH enzyme, and to be safer and less active against the hIMPDH2, which reflected its selectivity. Therefore, I. confusa underground parts and its isolated compounds can be used as excellent therapy to control gastric ulcers via eradication of H. pylori and exerting its anti-inflammatory potentials.
The PF of I. confusa underground parts exhibited high DPPH scavenging activity which was in accordance with its high TPC and TFC. The DPPH scavenging activity could be attributed to its total phenolic and flavonoid contents 58 . The observed anti-oxidant potential of the NPF could be attributed to the recently detected I. confusa xanthones and triterpenoids 29 . Xanthones and triterpenoids metabolites are well documented anti-oxidants 59,60 .
In recent years, irigenin, isolated from Belamcanda chinensis (Iridaceae) has been a hot research topic due to its important bioactivities. Irigenin controlled the metastatic progression in lung carcinoma cells 61 . It was recently proved to exhibit beneficial potentials in management of cardiac injuries. It alleviated doxorubicin (DOX)induced cardiotoxicity 62 and protected HUVECs (human umbilical vein endothelial cell lines) from angiotensin II-induced oxidative stress and apoptosis injury 63 . Irigenin exhibited inhibitory effects on prostaglandin E 2 and nitric oxide production in murine macrophage RAW 264.7 cells 64 .
Our results presented irigenin (P 3 ) as a new anti-H. pylori, COX, NO and HpIMPDH inhibitor beside being previously reported to significantly enhance TRAIL-regulated apoptosis in Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistant gastric cancer cells 65 . This highlights the promising expected results upon future testing of its in vivo potential in gastric ailments.
It is noteworthy to mention that, this is the first time to isolate irigenin from I. confusa by such classical chromatographic techniques. Thus, we herein present I. confusa as another natural source of irigenin other than Belamcanda chinensis (Iridaceae).
Besides irigenin, isolated in this study, the anti-H. pylori activity of I. confusa underground parts is attributed to its content of flavonoids, isoflavonoids, and xanthones which were recently reported in the PF 29 . Flavonoids and isoflavonoids react with superoxide anion, lipid peroxy, and hydroxyl radicals thus can protect the gastric mucosa against reactive O 2 species formed during the infection 50 . In addition, several natural flavonoids were reported to exhibit potent bactericidal potential against antibiotics resistant H. pylori strains 66 . Xanthones also are well documented to exhibit anti H. pylori activity 67,68 . Mangiferin xanthone reported in I. confusa PF 29 is a very potent gastroprotective 69,70 and anti inflammatory 71 compound.
It is worthy to note that, the H. pylori, COX-1, COX-2, and NO inhibitory potentials of irigenin (P 3 ), were much higher than that of PF from which it was isolated. Thus, fractionation and purification of I. confusa crude fractions caused significant increase in the aforementioned activities which may indicate a chance for isolation of more active constituents.
To the best of our knowledge, this is the first study that demonstrates the anti-H. pylori activity as well as COX-2 and IMPDH selective inhibitory activity of I. confusa underground parts and its isolated compounds. www.nature.com/scientificreports/ Novel strategies are urgently required to control H. pylori infection. Our findings recommend further studies on I. confusa bioactive metabolites and also suggest irigenin as an important lead for management of H. pylori infection after detailed in vivo and clinical studies.

Conclusion
Irigenin exhibited promising activity against HpIMPDH enzyme with low activity against human hIMPDH2 than clarithromycin, assuring its selectivity in addition to it's selective COX-2 inhibitory potential. This study scientifically validated the claimed anti-bacterial activity of I. confusa and has put strong focus on exploring traditional Chinese medicine for novel anti-H. pylori infections controlling drugs.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary  Information files).